The overall goal of this proposal is to generate, maintain, and distribute a new collection of engineered Drosophila melanogaster stocks that will allow versatile manipulation of genes for which there is currently no mutant allele available. Using cutting edge CRISPR technology, this collection or 'kit' will represent a powerful new set of tools that will benefit virtually all Drosophila geneticists, and it is likely that there will b a high demand for this resource for many years. Many of the most important advances in our understanding of human development have come from studies using Drosophila as an animal model system. Since many parallels exist between Drosophila and mammals in terms of the underlying molecular mechanisms controlling biological processes, knowledge gained from research in Drosophila can be either directly applied or readily adapted to understanding human biology. One key factor that sets Drosophila apart from other model systems is the huge wealth of genetic and molecular tools that have accumulated in the past 100 years of research. To study any given gene, two essential reagents are loss-of-function mutations and a tagged version of the gene. Currently available methods to obtain mutant or tagged alleles of genes cover only a portion of the genome or are inefficient, laborious, and time consuming. Indeed, there are more than 2000 genes in the fly genome that currently lack a mutant allele with no means of efficiently generating either a loss-of-function mutant or a tagged allele. To address this problem, we propose to generate a new targeted collection that will complement existing collections and offer the ultimate flexibility of downstream manipulation, such as generating both point and loss-of-function mutations, protein tagging, and gene expression profiling. We plan to split this project into two phases: Phase I will serve as a proof of principle and optimize the conditions for CRISPR- mediated targeting while Phase II will generate a collection targeting 2000 genes currently without a mutation. To achieve this goal, we will: Aim 1. Develop and optimize CRISPR-Cas9 mediated cassette targeting. Aim 2. Evaluate CRISPR-mediated cassette targeting across the genome. Success in Phase I will develop robust protocols for CRISPR-mediated cassette targeting and set the stage for expanding our effort to generate a complete collection during Phase II. Harnessing the benefits of new technologies, this collection will be another key addition to the Drosophila research tool kit. As a sign of the strong enthusiasm this proposal has received from many prominent Drosophila researchers, a set of 11 letters of support are included with this application.